Preparation of insulin from pancreas glands



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Patented May 6, 1952 PREPARATION OF INSUEINFR'OIM' PANGREAS GLANDS Loyal C. Maxwell, Chicago, and-.William P. Hinkel,

.Brookfield, 111., assignor site; Armour and. Conn.

parry, Ohicago Ill a. corporation. of lllinoisj.

NoD'rawing; Application April28}1950' Serial'aNo. 158,928

7 ".Claims.. (GREY- 7.5)

This invention; relates to the preparation-oi insulin from pancreas glands. This application constitutes a continuation-irr-partv of our copending. application, Serial No. 103,956, for Preparation of Insulin from Pancreas Glands,v now abandoned:

In the commercialmanufa cture ofinsulin; the practice has been to. extract the insulin from pancreasglands, using; acidified alcoholic solutions. employing hydrochloric acid: or. sulphuric acid. In suchextraction steps, the: pH of the extraction mixtureisnormally maintained: at about,2;0.. Thejpancreasglands are hashed and, in theusual commercial process, the insulin is extracted therefrom by using a: solvent consistinggof ethyl alcohol ,and hydrochl or-ic; acid, the alcohol being in a concentration of. approximately;65% to. 75% on the'basis-of the extraction volumeafter-equilibrium is attained; After adjustingthepH to" about" 2.0, the mixture is agitated for several hours and centrifuged, the meat solids being reextracted under the same conditions. The centrifugate liquors from both steps are then made alkaline, using; ammonia, and are filtered. The 'filtered extracts are concentrated/and further purified for insulin recovery.

The foregoing processgives. a, yield which amounts to only as-mall percentageof the insulin we. believed to be, actually contained in. the

glands Sincethe: amountrofinsulin available to, diabetic patientsiscritical, the-matter of yield is one of; great. importanca, and we set; about todiscover; somemethodwhich vWill...give greater yields of insulin in the extraction process v and further yields. of thefinal. finished productin crystallineor, amorphous condition for clinical administration. Wet-have .iound, that itzis. pos sible-to employ phosphoric acid in theextraction step and obtain asubstantially increased .yield .of insulin from; pancreasglandsa We. have. found. that. phosphoric. acid. has the unique property ofiormingsalts which are.relativel'y, insoluble in theextraction solvent. when the extracts are neutralizedlor. rendered. alkaline by. the. addition of a. suitable. base. This. unique feature permits. removal. of. the salts so-iormed in the. filtration. operation and; thereby. greatly aidsgin increasing the" amount. of, insulin recoveredby our process.-

Inv addition to. the. use of sulphuric, acid and hydrochloric acid;:as-- mentioned. above; organic acids, such:- as acetic; formic-1 and thelike, .may be employed. These; organic acids". have: to be usedin' such..quantitiwthat-their:usezissnotnon- .2. sidered. practicable.. 1 .rther, the presence of the large amount. ofzsoluble. salts of these acids are. undesirable in the; subsequent steps. of. the process.

We have. found that phosphoric. acid; unlike sulphuric,- acid hydrochloric acidIand-the above organic: acids,v has the. unique property of torming, insolublesalts,., thus. permittingthememoval of. thephosphoric. acidfiomthe process in an early. filtration:steptfollbwingthe extractionstep.

An object of [the inventionis' to provide .a-process; in. which phosphoric, acid. is employed in the extraction. step. A; further object of the: invention is tov provide a. process; in; whichphosphoric acid. is effectively employed within pH ranges found. effective. for the recovery. of insulin with a-high yield. thereofl. Yet another. object. is..to

providea process. inwhich lossesoffinsulinheretofore experienced in the concentration..of..tlie extract throughdistillation. in..vacuo is: elimihated. and; a. high recovery obtained following the" concentration or distillation.-- step;- Other specific. objects; and". advantages. will appear as the specification.proceeds.

The. pancreas. glands extracted, may-- be. from any source- Cattle, hogs-, or. other animals, fishes Whales, and the .lilie,. all. provide. sources of pancreas glands. which. may be. extractedlior the. recovery. of insulin.

- oneembodiment. ofiiour.process, ve, extract thehashedlpancreas;glands .in..an'.orga c solvent for insulin,.. sucli as. ethyl. alcohol andvinzatlie presence of? phosphoric. acid. Breferal51$...sufli cientphosphoric. acid-Z'is, employedto adjnstltliej pI-Fofthe extraction mixture to about. 3 .Th'e

' extraction mixture may then be filterediorcentrifixgedthremove the. suspended; solids. If. dc!

sired, the solids orlresiduej..mayj.be. reextracted undensulistantially ,theisamei conditions. andtlie filtrate. combinedfwith .tliefni stlfiltrate...

,After' the. extractionetea. we: prefer; tolaci'df a base; uch. s;aqueousammoniaptozbnhg:tnegpn 815; thereby: iiorming. 3 an; insoluble ammonium; salt. of *tlie phosiifioricj acid wliileiat.

t e. same; time precipitatingtj'inerti prote rial! Thesalhandthe inertiprotein m maythembe'rembv'ed'ffiyffiltiationi. Ah base for 'forminean.iiisoliiblesaltiiwitlith ,plifossphoric acidi-mayjlielemployedi For. examp1e, .-sodium hydroxidelmaytbe employedein lieu .ofalnmonia, We .prefers-tohemploy aweak base, .and aqueous ammonia-is particularly; efiective iniithe operation Thee-insulin zginz: thegalcoholicifiltiratestmayirbe recovered'zby.conventional-proceduresi 'Thfi filkfli mate line alcoholic filtrates are acidified and concentrated in vacuo at reduced temperatures and the insoluble lipoidal material removed by filtration. The insulin in the aqueous concentrate may be precipitated by the addition of sodium chloride, and the salt cake thus obtained may be further purified by solubilizing in water and precipitating the insulin at its iso-electric point. The recovered precipitated insulin may be solubilized and then crystallized after the addition thereto of zinc acetate or zinc chloride, to thereby obtain the zinc insulin salt.

Instead of the conventional purification method just described, it will be understood that other well-known purification methods leading to the preparation of finished amorphous or crystalline insulin compositions may be used.

We prefer to remove lipoidal material in the.

filtrate after the above filtration step in which the insoluble salt and inert protein is removed and prior to the purification of the filtrate to obtain insulin. After the filtration step, the filtrate obtained is adjusted by the addition of sulphuric acid (or some other acid) to a pH of 3-3.5 and concentrated under reduced pressure to an alcohol content of -25%, preferably about The lipoidal material, which then separates, may be removed by filtration or centrifugation, and the filtrate concentrated again under reduced pressure to the aqueous phase. There is a fur- .ther precipitation of lipoidal material, and this is then removed by filtration or centrifugation. The lipoidal material is preferably separated by partial removal of the alcohol under vacuum in a still. The temperature in this step is preferably kept as low as possible, which the still is maintained under reduced pressure. We have obtained good results by maintaining temperatures of 60-70 F. V In the precipitation of inert proteins, it is advantageous to adjust the pH into the range of 5.2 to about 9.5, with the preferred range being 5.5-8.5. Our best results have been obtained in the range of pH 8-8.5. The inert protein material is eifectively precipitated, and the removal I of this material, together with the insoluble salts of phosphoric acid, has a highly beneficial effect upon the subsequent operations.

The solvent for'the insulin may be any watermiscible organic solvent for insulin, such as ethyl alcohol, methyl alcohol, propyl alcohol, isopropyl alcohol, acetone, etc., or mixtures thereof. We prefer to use an aliphatic alcohol of less than [4 carbon atoms, and find ethyl alcohol the most desirable.

The concentration of the organic solvent in the extraction mixture should besufficiently high .to avoid substantial solubility of ,other substances,

such as the pancreatic enzymes, and should not be so high as to produce substantial insolubilization of insulin. We find that an alcohol concentration of to 85% by volume in the liquid present in the extraction mixture is satisfactory. Concentrations below 50% permit solubility of an undesirable amount of enzymes together with protein impurities, and concentrations above 85% produce a decreased yield of insulin. We prefer to 75% alcohol concentration.

When we refer to concentration in this specification and claims, we mean the over-all concentration in the extraction mixture on the basis of the total volume of liquid present and not the concentration of the solvent added to the process.

The temperature at which the extraction is conducted may vary throughout a wide range, but

we prefer to conduct the extraction step at temperatures between 0 and 15 C., and most suitably at about 10 C.

As above stated, in the recovery of insulin from pancreas glands, the hashed or macerated pancreatic tissue is extracted ordinarily in an acidified alcoholic solution containing 60% to 75% alcohol. The alcoholic extraction mixture ordinarily is acidified to a pH of about 2.0 with hydrochloric acid or sulphuric acid. Other workers more recently have reported using weak organic acids, such as acetic, formic, propionic and butyric acids, for this purpose.

We have shown that phosphoric acid is particularly suitable as an acidifying agent in the extraction of insulin from pancreatic tissue and that very considerably higher yields of insulin are obtained thereby than are obtained by the use of strong acids, such as hydrochloric or sulphuric acids. In the use of phosphoric acid for this purpose, we have found the maximal insulin recovery is obtained when the extraction mixture is acidified to a pH of about 2.5 or 3.0 and that lesser yields are obtained at a pH of above 4.0. We prefer to employ a pH of from 2.85 to 3.5. No substantial advantage is obtained by reducing the pH below 2.5 or raising it above 4.0.

We believe that the higher yields of insulin obtained with the use of phosphoric acid to be due to a combination of desirable properties possessed by this acid, which makes it particularly suited for use in the extraction of insulinfrom pancreatic tissue. This combination of desirable properties consists of the property of phosphoric acid of promoting maximum solubilization and extraction of insulin from pancreatic tissue without the necessity of using the excessively high acidity (pH 2.0) required when using hydrochloric or sulphuric acid, and, secondly, the property that the salts of phosphoric acid are relatively insoluble in the extraction solvent, so that the high salt concentration incurred in the extracts using hydrochloric, acetic or formic acid or other acids of this type, is avoided.

In the use of acetic, formic or hydrochloric acids in the extraction of insulin, it is necessary. after removing the gross pancreatic tissueafter extraction, to neutralize the acidity in order to clarify th extracts by filtration. This is usually performed by adjusting the pH of the extracts to pH 7-8.5 by the addition of aqueous ammonia. This alkalinization also causes the precipitation and allows the removal of undesirable protein'impurities which are precipitated in this pH range.

In the use of hydrochloric, acetic or formic acids, the adjustment of the pH of the extracts to 7.0-8.5 previous to filtration results in the introduction of very large amounts of soluble ammonium salts into the extracts. This is undesirable and does not occur when phosphoric acid is employed to acidify the extraction mixture, since the ammonium salts of phosphoric acid are relatively insoluble in the aqueous-alcoholic insulin extractant and are removed when the extracts are filtered after the pH has been adjusted toward neutrality or to the alkaline side with aqueous ammonia. This avoidance of the introduction of large quantities of soluble ammonium salts into the extracts contributes to higher insulin recovery, since the presence of such large amounts of salts. raises the boiling point of the extracts duringthe later concentration in the still to remove alcohol and in this way and otherwise is responsible for large losses of insulin.

The use of phosphoric acid also allows the removal oi other undesirable substances from the extractssuch-as iron, copper, or heaviermetals whose-salts may be present in small amounts. as salts-ofthe metals which mayhave been introduced as contaminationsin the 'reagentsizor materials or from the equipmentemployed in processing. It is known that the presence of such metallic ions causes. insu1 n.,lo5ses. The saltsof suchmetals with phosphoric acid are comparatiyely-nsolublein the extractant employed and removed he ih xtr e er eu r li e withv ammonia and filtered. Suchv metallic ions are o so completely removed when sulphuric, hydrochloric, ,fornoic or acetic. acids are used, ,eisalts of these acids are comparatively e inpthegextractant and'adjustment of the allgalineside does not servetto I.. e=m lete y s nce the. hy r x o f -t he metalsare somewhat more soluble than heir Pheee e e a t n er th io While, somawhatflarger amounts of phQSphoric acid. are. required'tojlower the pH of theextraction mixture proportionally than are required when ,strongacids} such a hydrochloric or sulphuric, areriised, this is compensated for by the fact that ma'ximal insulin extraction with phosphoricacid is obtained at apH range, of 3.0 to 4.0, while, withhydrochloric or sulphuric acids it is generally conoeded that a, pH of 2,0 orthereabouts is optimal. This represents ten times the 111 ion.concentrationnecessary with phosphoric acid. We believe that conducting the extraction at. the lower acidity of pHg3 004 results in better i a ie eee r t "W i refe nd th t wh n w k o n acids, H hasacetic or formic are used, that it is necessa tohuse sufficient, of these, acids to lower the pHof the extraction mixture to about 3 to 3.5 in order to obtain optimal insulin extraction. These.,-weal; organic acids have. acid dissociation oonstantoi 1;s to 1.75 1o. and three to ten times the weight, of such acids is required as, compared to.v the quantity of phosphoric acid required to lower the. extraction. mixture proportionally. Weconsider theuseof such weak acids impractical since, it considerably increases, the costiof processingdue to. the excessive quantities ofjqacidreq uired. Phosphoric acid is comparatively a stronger acid, having an acid. dissociation constant, of. 1.'1 1 0.- andfwefind this strength of acid particularly suitable for our purposes, and ade not findjthat acids with a dissociation conse t dissident.new a e s t b e ma preet e l l ed e p'nin u in e racti n-v "In adjusting the. pHof the centrifuged extracts upward ineorder to precipitate and remove undesirable proteins and insoluble salts of phosphoriciacid. we. prefer to use. a relatively weal; base, an'alkalinizin'g agent in order to avoid locahspotsof unduly high alkalinity such as may occurusing a strong base. We find that aqueous ammonia 'nonium hydroxide) is suitable and we prefer ,use. However, various weak or strong, bases can. be used although it isnecessary in order to avoid the introduction ofjlarge quantities ofsoluble salts into the extracts, to use a base which forms relatively insoluble salts with ehe rzhe e ee d heeei n n iro m n whe the pl iiaadjustedto pl lfiiito 9.5, In the; case 05 51 1 9 1? o sodium hy r xide, the precipitetien. f: inso u mmoni m. ots i m p o phatezstartsat about pH 5, the precipitation be.- ing: more complete with, increasing alkalinity. In. the. case of; phosphoric. acid, the precipitationv of? ammonium. phosphate: is" about two- 75;

' tissue. solids. by filtration witlioutgfirst ll;

thirds complete atrpH it anrraboutradfi completo at pI-I 8.0; e

Therefore; the precipitation of: insolubleasalts; and: 0t undesirable proteins can-be conducted bje tween pH 6.0 .and pH 9.5. We, howeven, prefer to; adjust the extractstto between pI-Ii .-'Z-.5.- anti-1&5, beforev filtration, since a. high alkalinity. :ise-nct, advantageous. and; a; minimum amount: ct-ithesalts-.-ot' phosphoricsacid. remains solublain range.-

In our preferredprocess, snificientgiotathseeliquid phosphoric .aci 'd,is: added to anaqueous-aicohohc. solution of between and-.95%.-.alcohpktcon'-. ten-t, ;;so thatwaften addition of the macerated pancreatic, tissue, the pHaof the extraction-mi it a. turedsnhcnt 3:0- and thee alcohol content; about 60%;):657 fter; equilibriumiseattai-ned. Ali on stirring:,-and: lowing;su=fiicien imcefore extrac tion-,; the meat I r olids are removed by centrituga-e tion and reextracted in 60% to 65% alcohot-with the: addition of suaffisient acids to maintainvthe same 1JH=and again centrifuged; The.- pH; ct the. centniiugedextracts isadjusted to pH Sad to: 9 ;5swith the additionof aqueous ammoniaand filtered through a filter press to i-remove preci-pitated insolubleisalts. and proteins-and other suspended material; Thetclcan-fi-ltcredtex-ttaots arateacidified to a pH- of: about 3.5 wi=th sulphuric. acid and further processed and concentratedanc l.v purified according to conventional and we llknown procedures. 7

In acidifying the a l coholio;extraction mixture, we do notfiind it advantageous to reduce the pH of the. extractionmixture-below pl-1 255, since we" obtain optiin a1 insulin yields -in-the r f about 3.0 to 315 and'we do notiapp re crease-the yield-by de inea e mate ac T e amount of insul-in extracted:'drops' -off ata, above 3.5 and the'yield drops oi'i liarply at pI-I;- above 4.0. The; desired rang is H; .2; and our preferred range is, 2 ,8 5 "3 5 While in the. foregoing example We here pointed out th'e'advantage ofusing ammonia with phosphoric acidin that the salts ob l centration of distillationwstepiitwilf beund Ste d h he m o a r atise t n maybe m t if d i ed; e iex m esoi he tr e s With ut h eeo am oni -w... specific examples .hereinaftcl; Y

The ueeofi. phe nho iciaeid; produces-cc dv n age eyer the. meet] e. am ne. Q chloric aci a su puuri ia iq Hydrocli a id eu eeeeew iea ndt e e of 'ee tissue. rendering itvery difiicul case rate t ing the extract. In alkalinizing tn extr large amount ofsolublee m i sai-ts troduced, which salts result in a considerable loss of insulin at later-stages in the process. Sulphuric acidis less efiectiveinthe extraction q eu and t. s n ee s tase uflieient ee d to-reduce the pH to 2. alo'w I iseens ent emen. f. a eee e s r eesi eut rious sulin; losse at; ete ze eeea. One. explanation. or-s chloss en hat the;sttonelyeaeidt atetiai enhances insulin fibrih formation-i during; the period when. the materiali-istheatedrandfbrought into: contact with the; walls;..of the-still... In con trast phosphoric. acid; is employed effectively, at a higher pH range.- so-thatthe-aduerseeffects upon insulin produced by thelow. pH conditions are avoided. Further, the highenpH range employed for extraction avoids-the. fprmationfof insulin fibrils.

There is a far less loss in insulin during the distillation step following extraction with phosphoric acid and this contributes greatly to the higher insulin yields obtained with our process. Unlike the hydrochloric acid or sulphuric acid extracts, the phosphoric acid extracts may be alkalinized with ammonia to produce insoluble ammonium salts which are removed in the filtration step prior to, the concentration of the extract by distillation. This results in lowering the salt content and ionic strength and improves insulin recovery by avoiding insulin loss due to fibril formation. J

'Umike tne hydrochloric acid, the use of phosphoric acid permits efiicient centrifugation and ready filtration of the extracts without the necessity of prior alkalinization. Unlike sulphuric acid;'the use of phosphoric acid results in ready soiubilization and extraction of insulin from panereatic tissue.

Phosphoric acid lacks the undesirable characteristics of sulphuric acid and hydrochloric acid in the above important processing steps while at the same time in actual practice the recovery of insulin through the use or phosphoric acid in the range described produces approximately 100% greater yield than that obtained in commercial processes where hydrochloric acid or sulphuric acid is employed.

. specific examples illustrating our process may be set out as follows:

Example I I In'commercial scale processing, 6090 pounds of frozen beef pancreas glands were bashed and macerated and extracted by stirring in 1860 gallons of 82% alcohol containing 153 liters of phosphoric acid (U. S. P.) After equilibrium was attained by stirring, the pH was 2.85 and the alcohol concentration about 65%. The pancreatic tissue insolubles were removed by centrifugation and reextracted under the same conditions. After centrifugation, the combined extracting liquid was alkalinized to pH 8.2 by the addition of aqueous ammonia and filtered. The filtered extracts were reacidified to pH 3.5 by the addition Of"H2SO4. Biological assay at this stage indicated the insulin content of the extracts to amount to 1800 I. U. per pound of glands processed. The reacidified extracts were then concentrated in the vacuum still to remove the alcohol and filtered to removelipoidal material. The 'aqueous'still concentrate at this stage was found to contain 1780 units" of insulin per pound of glands processed. The insulin was further purified by conventional procedures and converted to crystalline insulin. The yield of zinc insulin crystals obtained amounted to 1620 units per pound of pancreas glands processed.

Example .II

, 40'pounds of frozen pork pancreas glands were bashed and extracted by stirring with 45,500 cc. of 85% alcohol containing 925 cc. of phosphoric acid. The acidity of the extraction mixture was pH 3.0 and the alcohol concentration approximately 65% after equilibriumwas attained. The pancreatic meat solids removed were then reextracted by stirring in 45,000 cc. of 65% alcohol. The pH of the combined filtrates was raised to pH 8.5 by the addition of ammonia to precipitate the phosphoric acid salts and inert proteins. The solids were removed by filtration and sulphuric acid was then added to the filtrate to bring the pH to 3.5. The acidified extracts were then concentrated under reduced pressure to an alcohol concentration of 20% Lipoidal material was then removed by filtration and the insulin-containing filtrate biologically assayed for insulin activity. The biological assay showed the insulin recovered to be equivalent to 1615 international units for each pound of pancreas glands pro-v cessed.

Example 111 1,500 pounds of frozen beef pancreas glands were hashed and extracted by the use of phosphoric acid in the extraction step as described in Example I. The mixture was acidified to pH 3.5 with phosphoric acid. After extraction, the suspended solids were removed by filtration in a filter press. The insulin-containing filtrate was then adjusted to pH 8 by the addition of aqueous ammonia, and the precipitated ammonium salts of phosphoric acid and inert proteins were removed by filtration through a filter press. .The filtrate was then acidified to pH 3.5 with sulphuric acid. The mass of the lipoidal material was removed by distilling under reduced pressure to an alcohol content of approximately 20% followed by filtration and by further concentration under reduced pressure to the aqueous phase. The insulin in the filtrate was then recovered by the conventional procedure, using sodium chloride, etc. The yield of crystalline insulin amounted to 1400 international units per pound of original glands used.

Similar quantities of frozen pork pancreas glands were hashed and extracted as above de: scribed in Example III and with similar results. The yield of the final insulin product in the crystalline or amorphous form represented a large 1 increase over the yields obtained in processes em pounds of frozen beef pancreas glands were hashed into 118 liters of 82% alcohol containing 2200 cc. of phosphoric acid and extracted by stirring. The pH of thejextraction mixture was 3.1 and the alcohol concentration 65% after equilibrium was attained. The meat solids were removed by centrifugation and reextracted in 100 liters of 65% alcohol. The combined centrifuged extracts were adjusted to pH 8.2 with aqueous ammonia and filtered. The filtered extracts were then reacidified to pH 3.5 with the addition of sulphuric acid and concentrated in vacuo and filtered to remove lipoids. Biological assay of the aqueous concentrate showed the insulin recovered to amount to 187 6 units per pound of glands processed. The insulin in the aqueous concentrate was then salted out by the addition of sodium chloride and the insulin salt cake obtained solubilized in water, and the insulin precipitated at the iso-electric point. The insulin precipitate was then solubilized in water containing a buffer, and the insulin converted to zinc insulin crystals by the addition of zinc acetate. The yield of dried zinc insulin crystals recovered as determined by, weight and biological assay was 1526 units per pound of pancreas glands.

Example IV-A of 70% alcohol. The combined centrifuged extracts were adjusted to pH 7.6 by the addition of aqueous ammonia and filtered. The filtered extracts were reacidified to pH 3.5 by the addition of sulphuric acid and concentrated in vacuo and filtered to remove lipoids. Biological assay indicated the insulin content of the aqueous concentrate to be 1000 units per pound of pancreas glands processed. The insulin was salted out Example V 40 pounds of hashed frozen pork pancreas glands were extractedby stirring in 47 liters of 82% alcohol containing 800 cc. of phosphoric acid. The pH of the extraction mixture was 3.1 and the alcohol concentration 65 after equilibrium was attained. The meat residue was removed by centrifugation and then reextracted in 40'liters of 65% alcohol. The combined centrifugates were adjusted to pH 8.5 by the addition of aqueous ammonia and filtered.

A sample of the alcoholic insulin containing filtrates was removed and biologically assayed. The insulin content of the filtrate amounted to 2090 units per pound of pancreas processed.

The filtered extracts were then concentrated in vacuo and filtered to remove lipoids. The insulin content of the aqueous filtered concentrate as shown by biological assay was 1760 units per pound pancreas glands processed.

- Example V-A 40 pounds of hashd frozen pork pancreas consisting of a representative sample of the same lot of glands processed in Example V were hashed into 47 liters of 82% alcohol containing 9.2 pounds of liquid formic acid. The pH of the extraction mixture was 3.2 and the alcohol concentration approximately 65%. The meat residue was removed by centrifugation and reextracted in 40 liters of 65% alcohol. The com bined centrifuged extracts were then adjusted to pH 8.5 with aqueous ammoniaand filtered.

A sample of the filtered alcoholic extracts removed and biologically assayed showed the insulin content of the extracts to be 1680 units per pound of glands processed.

The filtered extracts were then reacidified and concentrated and filtered by a procedure identical to that employed in Example V. The yield of insulin in the aqueous concentrate by biological assay was 870 units per pound.

Example IV and IV-A, and Example V and V-A demonstrate the injurious effect of the presence of large amounts of soluble ammonium salts of such acids as hydrochloric acid and of formic acid, whose ammonium salts are not precipitated and removed in the extraction solvent employed. As observed in Example V and V-A, the insulin loss during concentration and filtration of the extracts is much less when phosphoric acid, whose ammonium salts are insoluble and are removed, is employed.

Example VI 40 pounds of hashed frozen beef pancreas glands was extracted by stirring in 47 liters of 91% alcohol containing 575 cc. of phosphoric acid. The pH of the extraction mixture was 3.5

and the alcohol concentration 70% after equilibrium was attained. The pancreatic tissue solids were removed by centrifugation and ertracted again under the same conditions. After centrifugation .the extracts were clarified by liltration before concentration in the vacuum still. Biological assay showed the filtered extracts to contain 1400 I. U. per pound. of glands processed.

The filtered extracts were then concentrated in the vacuum still without previous adjustment to the alkaline side.-;- The aqueous still concentrate from the extracts was treated withan immiscible solvent to remove lipoids and separated from the lipoidal fraction. Assay of the aqueous concentrate .showed the insulin present to amount to 1295 I. U. -per pound of glands processed. e: 4

Example VII 40 pounds of frozen'beef pancreas glands were macerated and extracted in 45,000 cc; of 82% alcohol containing 800 cc. of phosphoric acid. After stirring for one and one-half hours the pH of the extraction mixture was about 3.0 and the alcohol concentration 65% by volume.

The pancreatic tissue solids were removed by centrifugation and reextracted under the same conditions. The combined extracts were then filtered without alteration of the pH and assayed. The insulin content by biological assay at this stage amounted to 2200 I. U. per pound of glands.

The clarified extracts were then concentrated directly in the vacuum'still to'anv alcohol content of 20%. The concentrated extracts were then chilled and filtered to remove lipoids, and further concentrated to the aqueous stage. The insulin content by assay at this stage proved to be 1890 I. U. per pound of glands processed.

Example VI I-A 40 pounds of beef pancreas glands from the same lot as Example VII were processed in an identical manner with the procedure in Example VII with the exception that the extraction mixture was adiusted to pH 2.0 with sulphuric acid instead of with phosphoric acid. Assay showed the alcoholic extracts to contain 1540 I. U. per pound of glands and the still concentrate 920 I. U.

This example demonstrates not only the comparatively lesser solubilizing and extracting efiiciency of sulphuric acid as compared to phosphoric acid, but the much greater percentage of insulin lost in the concentration of "the alcoholic extracts to remove alcohol.

Our copending application, Serial No. 103,956, is in turn a continuation-in-part of our copending applications, Serial No. 709,090, No. 779,220,

abase to form insoluble salts of the phosphoric acid. Upon the removal of the salts, various optional procedures may be employed for recovering insulin from the extract. We have set out certain preferred steps, but it will be understood that such steps are not essential to the process as herein described and are set out merely as preferred procedure. After the phosphoric acid has been removed from the process in the form of ammonium salts of phosphoric acid, it is found that the resulting extract may be treated 'iegseums "inuch-more effectively former-recovery of insulin therefrom.

"While in 'Ith'e'foregoing "specification, certain steps. or the'process have been described in con- "siderable'detailfor the "purpose-of illustrating embodiments of the invention, it willbe under- Zstoodthat'such details maybe varied-widely by those skilled inzthe art without departing from thespiri'tof ourinven'tion.

We "claim:

2. A process for obtaining insiilin frompa'ncreas glands, comprisingsnbj'ecting said glands to extraiction:at apH of::about 2.5-3;5 with a r mixtureizoftphosphoric :aoidt iand arrorganic solwent foriinsulin' which is comnatible' with tphosphori'c =acid; separating :thesolid from the liqu'id portions, :andzirecoverin'g insulin :in the liquid portion.

' 3. A *processior obtaining insulin from pan- --creas-zglands, comprisingsubiecting said glands to extraction at a pH of about 25-41) with a mixture. of phosphoric acid and ethyl alcohol, and recovering-insulin irrthe extract.

:4. A ;process 'for" obtaining insulin .from pancreasiglands, comprising extracting said gglands atsa Zpl-rrdf: about 3.0 with .:phosphoric" acid \and an orgar-iivsoivent for insulin whlchl-isazcnmpati-ble "with phosphoric acid, separating the solid portionf-rom the liquid-portion, anilrecovering insulin in theliquidpoftion.

5. v A process for obtaining' insulin from pancreas glands, comprising "subjectingsaid" tg-lan'cls to extrac't-iomat a 15H- ofbetween 2.85 :and iiifi with a-mixture of phosphoric acid and ethyl aloohol; and recovering-insulin in the-extract'.

6. 'Inapr-ocess for obtaining-insulin irom pancreasglands, the steps of extracting 'Jsaid :g l'ands at a pH' oi about 2.5-4.0 with phosphoric acid and-an organic solvent for insulin which is compatible wiith phosphoric aoid, separating "the liquid from the solid portions, -and -cono'entrating the liquid by distillation.

7. In a process for obtainingiinsulin from pancreas glands, the steps of extractingsaid glands at a on "of between 2185 and 3'25 with-phosphoric acid andan organicsolventior 'instiIin which-is compatible with phosphoric acid, separating-the liquid. from the solid portions, and concentrating the liquid 'by distillation unclerva'cuum.

LOYAL C. WILLIAM P. HINKEL.

REFERENCES CITED The following references are of record in the file of this patent:

Somogy'i on the "fPreparation .of Insulin v in 

1. A PROCESS FOR OBTAINING INSULIN FROM PANCREAS GLANDS, COMPRISING SUBJECTING SAID GLANDS TO EXTRACTION AT A PH OF 2.5-4.0 WITH A MIXTURE OF PHOSPHORIC ACID AND AN ORGANIC SOLVENT FOR INSULIN WHICH IS COMPATIBLE WITH PHOSPHORIC ACID, AND RECOVERING INSULIN IN THE EXTRACT. 